This project is an investigation of the physiologic regulation of the digitalis-receptor (large subunit of NaK-ATPase) in the heart. Three aspects of this regulation will be studied: a) Control of the biosynthesis of the large subunit of NaK-ATPase by thyroid and adrenocortical hormones and by digitalis. The large subunit will be identified by phosphorylation and its turnover measured by double pulse labeling with 3H and 14C amino acids. Hormonal control of NaK-ATPase may represent a long-term adjustment of the cellular level of this enzyme (and possibly the digitalis-sensitivity of the heart) and may be the primary mechanism of action of some hormones. b) Relation between stimulation of NaK-ATPase by cyclic nucleotides and phosphorylation of membrane proteins by protein kinases. The stoichiometry of this process will be measured, the phosphorylated proteins separated by gel electrophoresis and their relation to NaK-ATPase and other transport systems examined. The possible dependence of these effects of cyclic nucleotides on the presence ("permissive action") of corticosteroids will be investigated. The effect of acute or chronic administration of digitalis on intracellular cyclic nucleotides will be measured in vivo and in isolated hearts and correlated with the "insulin-like" action of digitalis on glucose-transport. Through the effects of neurotransmitters on cyclic nucleotides, the autonomic nervous system may regulate the activity of NaK-ATPase indirectly. This regulation would be rapid but short-term. c) Biochemical determinants of the sensitivity to digitalis of cardiac NaK-ATPase. These postulated determinants are: amino acid composition of the large subunit of NaK-ATPase, lipid-composition of the sarcolemma and functional activity of sugar-binding site of the digitalis-receptor. These factors will be studied in cardiac sarcolemma prepared from digitalis-sensitive (adult guinea pig) and insensitive (rat, newborn guinea pig) animals. These factors may be responsible for phylogenetic and ontogenetic differences in digitalis-sensitivity.